Retention strength and selectivity of porous graphitized carbon columns Theoretical aspects and practical applications

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in

analyticalchemistry,

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1, 1995

[33] J. Franzen and R.H. Gabling, 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31-June 5, 1992,

p. 1009. S.A. McLuckey, G.J. Van Berkel, D.E. Goeringer and G. Glish. Anal. Chem., 66 (1994) 689A. [ 351 S.A. McLuckey, G.J. Van Berkel, D.E. Goeringer and G. Glish. Anal. Chem., 66 (1994) 737A. _ [36 1 S.A. Ruatt, J. Perel and J.F. Mahoney, 40thASMS [ 341

Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31-June 5, 1992,

p. 669. M.A. LaPack, J.C. Tou and C.G. Enke, Anal. [37 Chem.,

62 ( 1990)

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[38 M.A. LaPack, J.C. Tou and C.G. Enke, Anal. Chem., 63 (1991) 1631. [39 CL. Arthur and J. Pawliszyn, Anal. Chem., 62 (1990) 2145. 2. Zhang and J. Pawliszyn, Anal. Chem., 65 [40 (1993) 1187.

23

Dr. Gokhan Baykut is at Bruker-Franzen Analytik GmbH, Fahrenheitstr. 4,28359 Bremen, Germany. He received his Ph.D. in physical chemistry (ion cyclotron resonance, ICR, mass spectrometry) from University of Frankfurt in 1980. Between 1980 and 1982 he was at the University of Istanbul, and between 1983 and 1987 was a research associate at the University of Florida in Gainesville (ICR and ion photodissociation). After working as a senior scientist at lonspec Corporation in Irvine, California (1987-l 988), in 1989 he joined Bruker-Franzen Analytik GmbH, in Bremen, where he has been responsible for development of environmental sampling systems for mass spectrometry. He is now in charge of applications/development for Fourier transform ICR mass spectrometry. He also holds an associate professor degree of the University of Istanbul. His research interests include environmental mass spectrometry, Fourier transform ICR mass spectrometty, ion chemistry, and photochemistry.

Retention strength and selectivity of porous graphitized carbon columns Theoretical aspects and practical applications Esther Forgks

*, Tibor CserhSti

1. Introduction

Budapest, Hungary

Reversed-phase separation provides a versatile technique in high-performance liquid chromatography. Although silica, alumina, and zirconia are often used as support materials, graphitized carbon black has the advantages of being neutral and stable over a wide range of pH. Its poor mechanical stability has been improved by chemical and thermal processing to produce porous graphitized carbon supports. The properties of these supports, mechanisms of retention, and applications are discussed, along with correlations which can guide the choice of solvent combinations for typical separations. (0 1995

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Because of its versatility reversed-phase separation is widely used in high-performance liquid chromatography (HPLC) . Reversed-phase supports are generally prepared from silica by covalent bonding of various hydrophobic ligands to the surface silanol groups. However, the poor stability of silica towards alkali and the presence of free silanol groups on the surface after modification limit the application of silica-based supports in reversedphase HPLC. To increase the pH range of application, many other supports have been developed, including octadecyl-coated alumina and zirconia, and a variety of polymer-based supports. Active carbon has long been used as an adsorbent in classical liquid chromatography [ 1,2], *

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trendsinanalyticalchemistry,vol. 74, no. 7, 7995

24

Because of its high stability over a wide pH range and its neutrality much effort has been devoted to the development of graphitized carbon black (GTCB) supports and the assessment of their applications in gas chromatography and HPLC [ 31. Graphite is composed of layers of hexagonally-arranged, covalently-bonded carbon atoms, the layers themselves being held together by weak Van der Waals forces. To all intents and purposes, the graphitic sheets are giant aromatic molecules containing functional groups only at their peripheries. The adsorption of molecules on the non specific and nonporous surface of graphitized carbon black is mainly determined by the intermolecular dispersion forces between the solute molecules and the graphite surface [4]. Unfortunately, the otherwise attractive GTCB support was unsuitable for routine chromatographic work because it showed extremely poor mechanical stability. Several attempts have been made to treat GTCB in order to overcome this limitation. The best method was to heat GTCB at 900°C in a stream of nitrogen which had been saturated with benzene vapour at 30°C by passing it through a temperature-controlled bubbler. The partial pressure of benzene was 0.21 atm [ 5 1. The development of other graphitized carbon supports involved the deposition of pyrolytic carbon from the vapour phase of benzene followed by graphitization [ 5-71. Porous graphitized carbon (PGC) support was produced by impregnating a 7 pm spherical porous silica gel template with a melt of phenol and hexamine. The impregnated materials were heated gradually to 150°C to form phenol-formaldehyde resin within the pores of the silica gel. The resin was then carbonized by heating slowly to 900°C. After dissolution of the silica template with aqueous KOH, the carbon was heated to 2500°C in an oxygenargon atmosphere [ 8-111. The following parameters characterize the PGC support [ 121: 0 Sufficient hardness to withstand high pressures (48 MPa); 0 a well-defined, reproducible and stable surface that shows no change during chromatographic work or storage; 0 a specific surface area in the range 150-200 m*/g, to give adequate retention of solutes and maintain a reasonably linear sample capacity over a large concentration range; 0 a mean pore diameter of 2 1Onm and the absence of micropores to ensure rapid mass transfer of solutes into and out of the particles;

uniform surface energy to give a linear adsorption isotherm. Because PGC is practically inert to acids and bases, eluents of any pH can be used. Many experiments indicate that the apparent hydrophobicity of the surface of the PGC support is higher than other traditional reversed-phase supports such as octadecylsilica (ODS) or phenyl-bonded silica [ 13,141. This implies that a higher percentage of organic modifier is required for elution of a given solute from a PGC column, the increase generally being 2040 vol.%. However, in addition to its extremely hydrophobic nature, PGC is also unique in possessing a conduction band of delocalized electrons which can considerably modify solute retention. Using selected solutes as examples, an attempt has been made to establish the eluotropic strengths (9) of some organic solvents on PGC [ 151. The results showed that the actual value of 9 for a particular solvent depends greatly on the chemical character of the solute or solute group used to determine it, and that the differences in the eluotropic strength are usually small. For the majority of solutes, methanol and acetonitrile show low elustrengths while otropic dioxane and tetrahydrofuran have higher values. It was further established that ethyl acetate, dichloromethane, butyl chloride and hexane have eluotropic strengths that vary most strongly with the solute. It was concluded that an eluotropic series comparable to those for silica or alumina is difficult, if not impossible, to establish for PGC supports [ 151. 0

2. Retention behaviour of PGC column The retention mechanisms of PGC supports and the role of various hydrophobic and hydrophilic (electronic) forces in the interaction of solutes with the PGC surface have been vigorously discussed, although the conclusions are sometimes contradictory. The earlier studies considered PGC to be a ‘pure’ reversed-phase support because it has no free silanol groups which are not covered by the hydrophobic ligand, or other polar adsorptive centres on the surface. A study of the retention behaviour of 24 substituted aromatic compounds in non-polar eluents concluded that direct electronic interactions can arise by mixing of the HOMO or LUMO orbitals of the solute with the surface electrons of PGC or by charge transfer between the solute and PGC. It was demonstrated by a statistical

trends in analytical chemistry,

vol. 14, no. 7, 1995

25

A

B

= )

J-Yminutes

Fig. 1. Separation of aniline derivatives on porous graphitized carbon column. Eluent, methanol-water (955, v/v); flow-rate, 1 ml/min; detection, 254 nm. A = aniline, B = 2-chloroaniline, C = 3,5dichloroaniline, D = 2,4-dichloroaniline (unpublished results of the authors).

I

minutes

sdoo

Fig. 2. Separation of (A) 1 -ethyl-2-phenyl-3-o-nitrobenzoyl-4-methyl-and (B) 1-ethyl-2-phenyl-3-pnitrobenzoyl-4-methylbarbiturate, on porous graphitized carbon column. Eluent, methanol-water (80:20, v/v); flow-rate, 0.8 mllmin; detection, 230 nm (unpublished results of the authors).

1A approach that the PGC primarily behaves as an electron-pair acceptor for substituted aromatic solutes which are capable of n-electron donation under non-polar conditions. The retention order of the solutes closely followed the basicity of their loneelectron pair [ 16,171. These data indicate that physico-chemical parameters other than molecular hydrophobicity may influence solute retention on PGC. Other studies, using a different set of solutes, reached similar conclusions. Quantative structureretention relationship (QSAR) calculations were used to elucidate the retention mechanisms of PGC. The retention of 29 ring-substituted phenols [ IS], 23 ring-substituted anilines [ 191, and 45 barbituric acid derivatives [20] were determined in unbuffered acetonitrile-water (except for the barbiturates) and methanol-water eluent systems (see Figs. I-3), and the relationship between the log k’ and the concentration of organic modifier (C, vol.%) was calculated separately for each solute and eluent system using Eq. 1. log k’ = log k’()- bC

(1)

minutes

Fig. 3. Separation of phenol derivatives on porous graphitizedcarbon column. Eluent, acetonitrile-water (80:20, v/v); flow-rate, 1 ml/min; detection, 254 nm. A = 2,3-dimethoxyphenol, B = 2,6-di-tek-butylphenol, C= 2,6-dibromophenol, D = 2,4-dibromophenol (unpublished results of the authors).

trends in analytical chemistry, vol. 14, no. 7, 1995

The significant relationships between the retention parameters (log kfO and b values in Eq. 1) and the electron-withdrawing power, proton-donor capacity, and steric effects of substituents proved that the retention behaviour of the above compounds is mainly governed by these polarity parameters. The results suggest that the graphite surface is sensitive to changes in the solute electron-density caused by the electron donating and withdrawing abilities of the solute’s substituents and the number and position of the electron-dense bonds. Further, the PGC is highly sensitive to steric changes that disturb the electron density of the solute molecules and their interactions with the graphite surface. The position of substituents on the ring of the solute determines how it can approach and interact with the PGC surface. The lipophilicity of the compounds mentioned above did not significantly affect the retention parameters, log kfOand b. This finding, and the fact that the retention orders of solutes on PGC and ODS columns was not correlated emphasizes the marked differences between the retention characteristics of PGC and ODS columns, even though the eluents used with PGC columns are typical reversed-phase eluents which are also generally used with ODS columns [ 13,141.

3. Applications

of PGC columns

As has been mentioned previously, in planning separations on PGC columns one has to take into consideration the fact that PGC is more hydrophobic than ODS, so a higher ratio of organic modifier is needed for elution of given solutes. However, the electronic interactions between the surface of a PGC support and the solute may markedly modify the retention, and these interactions must also be considered. 3.7. Pharmaceuticals The neutral surface of PGC makes it specially suitable for the separation of basic solutes, as was demonstrated by the separation of remoxipride and FLA-98 1, two potential neuroleptic agents [ 211. Two methods were compared: separation with ion supression at pH 10 (50% acetonitrile in 0.1% ammonium hydroxide) and by ion pairing with TFA (50% acetonitrile in 0.1% TFA) . The superiority of the TFA mobile phase system was established, remoxipride and FLA-98 1 being eluted with convenient retention times (remoxipride ca. 3 min,

FLA-98 1 ca. 12 min) . With ion supression, excessive retention was observed for both compounds (remoxipride ca. 18 min, FLA-98 1< 60 min) . The purification of a thiconazole derivative with marked anti-hypoxia activity was not adequate on an ODS column. However, PGC and tetrahydrofuran-water (7:3 v/v) with 1% ammonia as eluent allowed the thioconazole derivative (retention time 3.5 min) to be separated from its impurities (at 6 and 9 min) [22]. Various substituted propargylamine derivatives show promise as monoamine oxidase inhibitory drugs. The retention times of seventeen monoamine oxidase inhibitory drugs were determined on a PGC column using unbuffered methanol-water and acetonitrile-water mixtures at various concentrations and the log k10and b values were calculated according to Eq. 1. The difference between the retention times for any pair of solutes, with any concentration of organic modifier, can be calculated as: t, _t2=tO(10al+blC_

lOaz+bzC)

(2)

where a and b are the intercept (log k’,) and slope values for solutes 1 and 2 at organic phase concentration C. The eluent composition corresponding to the maximum retention time difference can also be calculated: the first derivative of Eq. 2, must be zero, and the organic phase concentration is expressed accordingly [ 231: C= (a, -ua2+log

b,lb2)l(b2-bl)

The same chromatographic parameters (log k’,, and b values) for 45 barbituric acid derivatives on a PGC column with unbuffered methanol-water eluent mixtures were also published [ 201. 3.2. Xenobiotics We indicated earlier that retention parameters (retention time, log k’,, and b values) have been published for 29 ring-substituted phenol- and 23 ring-substituted aniline derivatives, determined in unbuffered methanol-water and acetonitrile-water eluent mixtures. These derivatives elute as sharp symmetric peaks and their retention order does not follow their lipophilicity order. The retention parameters of both aniline and phenol derivatives were different in eluents containing methanol and acetonitrile, proving the different selectivity of these organic modifiers. The published retention data make it possible to select the optimal eluent systems for the separation of phenol and aniline deriv-

trends in analyficalchemistty, vol. 14, no. 7, 1995

atives as described above (see Eqs. 2 and 3) [ l&24-26]. The separation capacity of PGC columns for the important agrochemicals, the chlorophenoxyacetic acid congeners, has also been explored using a dioxane-water mobile phase, without additives and with added sodium acetate, acetic acid or lithium chloride. The results indicated that acetic acid had the greatest effect on the retention, emphasizing the considerable role of the degree of dissociation on the retention on a PGC column. Retention parameters (log k10and b) have been given for each chlorophenoxy-acetic acid congener and eluent system [ 271.

4. Resolution of enantiomers Separation of the enantiomers of racemic drugs is very important because, in many instances, the isomeric forms show very different biological efficiency and toxicity. Diastereomeric pairs of the cephalosporin antibiotic Axetil E47 (A -3 isomers) and by-products from the synthesis (A -2 isomers and anti-methoxy isomers) have been successfully resolved on PGC with the eluent mixture acetoni(35:20:35: 10). trile-water-methanol-dioxane (The retention times of the active A -3 isomers were 4 and 7 min, and A-2 isomers 6 and 6.5 min, and anti-methoxy isomers 10 and 17 min) [ 281. The resolution of the enantiomers of two closely related benzodiazepine derivatives, oxazepam and lorazepam, was also achieved on a PGC column with /3-cyclodextrin as a mobile phase additive. Interestingly, the addition of 3 mM P-cyclodextrin to the mobile phase of acetonitrile-0.005 A4 phosphate buffer (pH 12) yielded an optical separation of both the oxazepam and lorazepam enantiomers. Lower and higher P-cyclodextrin concentrations were less effective [ 291. Not only cyclodextrins but also other chiral discriminators have been used for enantiomer separation on PGC columns. The separation of the optical isomers of a whole series of amino-alcohol P-blockers was achieved by adding carbobenzoxyglycil-L-proline (2.5 rnM) to the mobile phase (dichloromethane containing 0.4 mM triethylamine). The modification of the PGC surface with a monolayer of tris(phenylcarbamoyl)cellulose also resulted in a stationary phase suitable for the separation of heterocyclic N-containing sulfoxide enantiomers [ 301.

27

5. Other applications Inorganic anions possess no hydrophobic groups and are therefore not retained on silica-based reversed phase supports. On ionization in aqueous solution, however, they possess lone pair electrons which can interact elecronically with the conduction band of 7~ electrons on the PGC, leading to retention. This principle has been exploited for the separation of the 0x0 anions of technetium and rhenium on PGC. These two highly hydrophilic anions were eluted at the void volume on silica- or polymer-based reversed-phase supports, but were strongly retained on PGC when distilled water was used as the mobile phase. The retention was not affected by gradient elution with an acetonitrile content in the mobile phase increasing from 5 to 100%. However, on addition of 1% trifluoroacetic acid (TFA) the anions were eluted as sharp peaks and the capacity ratios (k’) increased with decreasing TFA concentrations [ 2 11. The separation of the 0x0 anions of technetium and rhenium suggested the possibility of employing the same technique for organic anions. The quantitative determination of oxalic acid in urine is of considerable clinical importance, but its separation from the other organic constituents in urine cannot be successfully carried out on ODS supports. The sample preparation and pre-purification steps were the same as with an ODS column, with the urine samples first being acidified with TFA or HCl to dissolve any oxalic acid present as its calcium or magnesium salts. The solution was then passed through a silica-based Cig precolumn, on which most of the hydrophobic contaminants were retained. Oxalic acid which was nor retained on the Cl* support was successfully separated from the endogenous impurities with 0.08% aqueous TFA solution on a PGC column, with a retention time of 5 min. To our knowledge this was the first use of a Cl8 precolumn coupled to a PGC column, and considerably increased the efficiency of the separation. The same clean-up procedure can be used for the analysis of creatine and creatinine in urine because they are not retained on the Cl8 precolumn in aqueous TFA solution. The optimal eluent system for the simultaneous separation of urinary creatine and creatinine was 3% (v/v) acetonitrile in 0.1% TFA (creatine, 3 min; creatinine, 7 min)

[211. The extremely hydrophobic surface of PGC has led to difficulty in eluting many hydrophobic sol-

28

utes, particularly those with planar structures which fit well on the flat hexagonal structures on the surface of PGC. Howewer, the surface can be modified by adsorption of surfactants, hydrophobic molecules, or high-molecular-mass polymers. These modifications result in lower retention capacity and changed retention characteristics of the PGC and allow the separation of compounds which would otherwise be totally retained. For example, it has been shown that a monolayer of Tween 80 (polyoxyethylenesorbitol stearate) adsorbed onto the surface of PGC reduces the log k’ values of hydrophobic molecules by 15-20%. A PGC coated with a hydrophilic polymer (polyvinyl alcohol, PVA) has been used for size-exclusion chromatography. Proteins, including thyroglobulin, transferrin, ovoalbumin, myoglobin and cytochrome c, were eluted in roughly reverse order of molecular mass from a PGC column coated with PVA multilayers ( 10 pmol m-* of OH) using 20 rniWphosphate buffer as eluent [ 151.

6. Conclusions Porous graphitized carbon (PGC) supports show unique retention characteristics. Separations on PGC columns use typical reversed-phase eluents (water, and organic modifiers miscible with water). However, the retention order of solutes generally does not follow their order of hydrophobicity. Molecular hydrophobicity influences but does not determine the elution order of any set of solutes. It has been proven that electrostatic interactions may occur between the planar ring substructures of solutes and the hexagonal graphite molecules on the PGC surface. These interactions have a considerable impact on the retention capacity and selectivity of PGC. Porous graphitized carbon supports make possible the separation of many solutes (of polar or apolar character), and seems to be specially suitable for the separation of positional isomers containing polar substructures. Because of the inert surface, PGC columns can be used over the complete pH range without deterioration of column efficiency and is therefore well suited for the separation of both basic and acidic solutes. The influence of buffering of the eluent on the retention is not clearly understood. Sometimes good separations of solutes with highly dissociable polar groups have been achieved, but in other cases the pH of the eluent had a considerable effect on the

trends in analytical chemistry, vol. 14, no. 1, 1995

retention. As with other reversed-phase supports, TFA has proven to be an excellent general purpose mobile phase additive for the separation of anions and other electron-rich species, and can be used as an ion-pairing agent for cations. Enantiomer separations can also be carried out on PGC columns using slight modifications of the separation methods developed for traditional supports.

References [l] A.V. Kiselev, Ad. Chromarogr., 4 (1967) 193196. [ 21 M.J. Telepchak, Chromatographia, 6 ( 1976) 234-242. [ 31 A.V. Kiselev and Ya.1. Yashin, Zh. Fiz. Khim., 40 (1970) 1272-1285. [4] W. Engelhard, J. Pijrschmann and T. Welsch, Chromatographia, 30 (1990) 537-543. [5] H. Cohn, C. Eon and G. Guiochon, J. Chromatogr., 119 ( 1976) 41-57. [6] H. Colin, C. Eon and G. Guiochon, J. Chromatogr., 122 ( 1976) 223-242. [ 71 H. Colin and G. Guiochon, J. Chromatogr., 137 (1977) 19-25. [ 81 K. Unger, P. Roumeletios, H. Miiller and H. Goetz, J. Chromatogr., 202 (1980) 3-14. [ 91 M.T. Gilbert, J.H. Knox and B. Kaur, Chromatographia, 16 (1982) 138-146. lo] J.H. Knox, K. Unger and H. Mtiller, J. Liq. Chromatogr., 6 (1983) l-32.

111 J.H. Knox, B. Kaur and G.R. Millward , J. Chromatogr., 352 (1986) 3-25.

[ 121 B. Kaur, LC.GC ht., 3 (1990) 41-48. [ 131 E. Forgacs, T. Cserhati and B. Chromatographia, 36 (1993) [ 141

Bordas,

19-26.

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115-122.

[ 151

J.H. Knox and B. Kaur, in P.R. Brown and R.A. Hartwick (Editors), High Per$ormance Liquid Chromatography, Wiley, New York, 1989, pp. 189-222. [ 161 R. Kaliszan, K. Osmialowsky, B. Bassler and R. Hartwick, J. Chromatogr., 499 (1990) 333-344. [ 171 B. Bassler, R. Kaliszan and R. Hartwick, J. Chromatogr., 461 (1989) 139-147. [ 181 E. Forgacs, K. Valko and T. Cserhati, J. Liq. Chromatogr., [ 191

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3457-3473.

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(1992) 356-360. [20] E. Forgacs and T. Cserhati, J. Pharm. Biomed. Anal., 10 (1992) 861-865. [21] G. GuandC.K.Lim,J. Chromatogr.,515 (1990)

183-192. [22] J.C. Berridge, J. Chromatogr., 449 (1988) 317321.

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u31 E. Forghcs, K. Valk6 and T. CserhBti, J. Chromatogr., 631 (1993) 207-213. ~241 E. ForgBcs, T. Cserhati and K. Valkb, J. Chromatogr., 592 (1992) 75-83. v51 T. Cserhhti and H.E. Hauck, J. Chromatogr., 514 ( 1990) 45-55. E. [261 Forghcs and T. CserhBti, J. Chromatogr., 600 ( 1992) 43-49. [271 T. Cserhhti, and E. ForgBcs, J. Chromatogr., 643 (1993) 331-336.

[29] A.Fell,T.A.G.Noctor,J.E.

[281N.W. Smith and D. Brennan, Poster presented at

Esther Forgks and Tibor Cserha’ti are at the Central Research Institute for Chemistry, Hungarian Academy of Sciences, P. 0. Box 17, H1525, Budapest, Hungary,

the 12th Int. Chromatography,

Symp. on Column Liquid Washington, DC, June 19-24,

1988.

MamaandB.J.Clark,

J. Chromatogr., 434 ( 1988) 377-384. [ 301 J. Dolphin, Lab. Pratt., 38 ( 1989) 7 l-82.

Micellar enhanced spectrophotometric determination of organic species J.S. Esteve-Romero * CastelId, Spain

E.F. Simb-Alfonso, M.C. GarciaAlvarez-Coque, G. Ramis-Ramos Valkncia, Spain The use of surfactants to enhance spectrophotometric procedures for the determination of organic species is critically reviewed. Special emphasis is placed on the effects of sutfactants on chromogenic derivatization The following topics are reactions. addressed: modification of acid-base and solubility equilibria, spectral changes, modification of reaction rates, and applications in equilibrium-and kinetic-basedanalyticalprocedures. Applications to flow injection procedures are also briefly reviewed.

been paid to other organized media such as microemulsions or liquid crystals. In the presence of surfactants, equilibrium, kinetic and spectral properties can be modified, and this has been used to improve the characteristics of analytical procedures. The changes are observed over a wide range of surfactant concentrations, below and above the critical micelle concentration (cmc) . Surfactants can induce favourable shifts in equilibrium constants and spectral properties, inhibit undesirable reactions, such as hydrolysis and photolysis, stabilize reaction intermediates, co-solubilize nonpolar and polar samples, derivatization reagents and products, and speed up reactions by means of micellar catalysis. Here we discuss these effects in relation to the enhancement of spectrophotometric procedures for the determination of organic species. 2. Modification of physical and physicochemical properties

1. Introduction The use of surfactants in various areas of analytical chemistry has attracted much interest in the last two decades. Normal or reversed micelles are most frequently used, and much less attention has * Corresponding author.

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Organic ions and molecules can bind the surfactant assemblies by both electrostatic and hydrophobic interactions. Thus, in a water-continuous micellar solution the non-polar parts of the solute molecule interact hydrophobically with the exposed hydrocarbon chains of the surfactant. If both solute and surfactant are ions or zwitterions, electrostatic interaction is also produced. The con01659936/95/$09.50